Self vs nonself in tissue assembly: Correlated changes in recognition behavior and tissue cohesiveness

Vertebrate embryonic cell populations of unlike kind, when combined in vitro, typically spread around and sort out from one another in combination-specific patterns, whereas like cell populations merely coalesce. These differing responses to self and nonself constitute one form of morphogenetic self-recognition behavior. Prolonged shaker-flask culturing and dissociation and reaggregation of embryonic chick heart tissue were both previously shown to reverse the tissue's spreading behavior with liver. Here, we show that these treatments simultaneously initiate, in heart tissue, a “foreign” spreading reaction toward untreated heart. Moreover, the direction of this heart-heart spreading can be deduced from the change in direction of heart-liver spreading. This suggests that certain properties of heart tissue participate in the determination of both the foreign- and the self-recognition behaviors studied here. The differential adhesion hypothesis postulates that these properties are the intensities of tissue cohesion, with less cohesive tissues enveloping more cohesive ones. If so, our observations imply that heart fragments precultured $\text{1}\text{2}$ day should be more cohesive than $\text{1}\text{2}\text{-}\text{day}$ precultured heart reaggregates, but less cohesive than heart fragments precultured $\text{2}\text{1}\text{2}$ days. With our centrifugation assay, in which relative tissue cohesiveness is assessed by the relative roundness of centrifuged aggregates at shape equilibrium, we confirm this prediction.     

The induction of cytochrome P-448 dependent benzo(a)pyrene hydroxylase in Saccharomycescerevisiae

When grown in high concentrations of glucose, the yeast $\text{Saccharomyces}\text{cerevisiae}$ produces a microsomal cytochrome P-450 monooxygenase system which is capable of hydroxylating benzo(a)pyrene. The addition of benzo(a)pyrene to the yeast during growth causes only a small increase in cytochrome P-448 levels but results in a dramatic improvement in the apparent kinetics of benzo(a)pyrene hydroxylation as measured by a decrease in the Michaelis constant and an increase in maximal velocity. Dimethylnitrosamine, phenobarbital and 3-methylcholanthrene also induce this enzyme to various degrees. Yeast pretreatment with β-naphthoflavone did not affect this enzyme, yet pretreatment with lanosterol resulted in a decreased affinity for benzo(a)pyrene. The addition of benzo(a)pyrene to yeast growing at low glucose concentration does not induce cytochrome P-448. The implications of these findings with regard to the presence of multiple forms of cytochromes $\text{P-448}\text{P-450}$ in yeast are briefly discussed.     

Evaluation of immobilized cytochrome P-448 from Saccharomyces cerevisiae using permeabilized whole cell, microsomal fraction and highly purified reconstituted forms, with benzopyrene-3-monooxygenase activity

Cytochrome P-448 from Saccharomyces cerevisiae in permeabilized whole cell, microsomal fraction and in a highly purified reconstituted benzopyrene-3-monooxygenase (EC 1.14.14.1) system have been immobilized on various supports. Calcium alginate was found to be especially useful and the kinetics of hydroxylation were close to that of the free enzyme system with all three forms of enzyme, even with permeabilized whole yeast cells (V _max of 664 pmol 3-hydroxybenzo(a)pyrene produced per h per nmol cytochrome P-448 compared with 1000 for free highly purified reconstituted enzyme system). Only the highly purified reconstituted form was successfully immobilized by BrCN-activated Sepharose-4B or by acrylamide. Both of these supports stabilized the highly purified reconstituted cytochrome P-448 benzopyrene-3-monooxygenase activity in prolonged storage at 4°C. Applications for various immobilized enzymes and cells are assessed.     

The use of electromyography for the assessment of sense of muscular effort: a test–retest reliability study

This study sought to examine the test–retest reliability to measure sense of muscular effort with electromyography (EMG). The EMG activity of the tibialis anterior muscle from 23 participants was recorded. Targets of EMG amplitudes produced at 10 and 20% of the maximum voluntary contraction (MVC) were calculated. Participants matched the target EMG level with and without visual feedback (FB). With NFB, the reliability was good to excellent when errors were represented as the average standard deviation (SD) of the error from the target (ICC_1,2 = 0.75 and 0.69 for 10 and 20% targets, respectively). Also, reliability was good when errors were presented as the average SD as a percentage of the MVC EMG (intraclass correlation coefficient (ICC_1,2) = 0.67 and 0.66, respectively, for 10 and 20% targets). Standard deviation around the target was the most reliable method to represent the error. This approach could be used as a simple cost-effective method to assess the sense of muscular effort.     

Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

ABSTRACT: BACKGROUND: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. RESULTS: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. CONCLUSIONS: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.     

Filamentous bacterial viruses. XI. Molecular architecture of the class II (Pf1, Xf) virion

The diffraction patterns of the Pf 1 and Xf strains of filamentous bacterial viruses (class II) can be interpreted in terms of a simple helix of protein subunits with 15Åpitch, having 22 units in five turns. The protein subunits are each elongated in an axial direction, and also slope radially, so as to overlap each other, giving an arrangement of subunits reminiscent of scales on a fish. The protein helix forms a tube with inner diameter about 20Åand outer diameter about 60Å. The single-stranded circular DNA is contained within this tube, with two DNA strands running the length of the tube.The diffraction patterns of fd, If 1 and IKe (class I) can be interpreted in terms of a perturbed version of the class II simple helix.     

Toxoplasmal and Toxocaral Infections: A Clinical Investigation into Their Relationship

An investigation was carried out to determine whether a relationship existed between infections with Toxoplasma gondii and Toxocara canis. Antibodies were sought by the toxoplasma dye test and the toxocara skin sensitivity test. Sixty-seven patients were examined in the United Kingdom; 38 had positive toxoplasma dye tests, two of these being toxocara-positive; and 29 had negative dye tests, six of them being toxocara-positive. From the total of eight toxocarapositive patients antibodies to toxoplasma were detected in two, an incidence no greater than that expected for a normal population.Sera from 60 toxocara-positive African patients were examined; 20 possessed antibodies to Toxoplasma. Analysis according to age group or geographical location indicated that this incidence was no greater than that expected for patients without evidence of toxocaral infection.This study therefore showed no causal or clinically important relationship between toxoplasmal and toxocaral infections.     

STICCS Reveals Matrix-Dependent Adhesion Slipping and Gripping in?Migrating Cells

Two-color spatio-temporal image cross-correlation spectroscopy (STICCS) is a new, to our knowledge, image analysis method that calculates space-time autocorrelation and cross-correlation functions from fluorescence intensity fluctuations. STICCS generates cellular flow and diffusion maps that reveal interactions and cotransport of two distinct molecular species labeled with different fluorophores. Here we use computer simulations to map the capabilities and limitations of STICCS for measurements in complex heterogeneous environments containing micro- and macrostructures. We then use STICCS to analyze the co-flux of adhesion components in migrating cells imaged using total internal reflection fluorescence microscopy. The data reveal a robust, time-dependent co-fluxing of certain integrins and paxillin in adhesions in protrusions when they pause, and in adhesions that are sliding and disassembling, demonstrating that the molecules in these adhesions move as a complex. In these regions, both α6β1- or αLβ2-integrins, expressed in CHO.B2 cells, co-flux with paxillin; an analogous cotransport was seen for α6β1-integrin and α-actinin in U2OS. This contrasts with the behavior of the α5β1-integrin and paxillin, which do not co-flux. Our results clearly show that integrins can move in complexes with adhesion proteins in protrusions that are retracting.